I. Field of the Invention
The present invention relates generally to the diagnosis and treatment of Ehrlichia infection. In particular, the invention is related to p120/p140 immunoreactive peptides derived from Ehrlichia proteins, and the use of such peptides in the detection of Ehrlichia infection in humans and animals.
II. Background and Description of Related Art
Ehrlichia chaffeensis and Ehrlichia canis are tick-transmitted, obligately intracellular bacterium that cause monocytrotropic ehrlichiosis, an emerging life-threatening disease in humans and a mild to severe disease in wild and domestic canids. A number of studies have demonstrated that antibodies play an essential role in immunity against Ehrlichial pathogens (Feng and Walker, 2004; Winslow et al., 2003; Winslow et al., 2000; Yager et al., 2005). However, only a small subset of E. chaffeensis and E. canis proteins react strongly with antibodies in sera from infected humans or dogs, and thus are considered to be major immunoreactive proteins (Chen et al., 1997; Chen et al., 1994; McBride et al., 2003; Rikihisa et al., 1994). Molecularly characterized major immunoreactive proteins of E. chaffeensis and E. canis include four protein ortholog pairs (p200/p200, p120/p140, p47/p36, and VLPT/p19, respectively) (Doyle et al., 2006; Luo et al., 2008; McBride et al., 2003; McBride et al., 2007; McBride et al., 2000; Nethery et al., 2007). Three of these ortholog pairs (p120/p140, p47/p36, and VLPT/p19) have acidic serine-rich tandem repeats (TRs), and continuous species-specific epitopes have been identified in the TRs of p47/p36 and VLPT/p19 (Doyle et al., 2006; Luo et al., 2008; McBride et al., 2007; McBride et al., 2000).
The p120 is differentially expressed by dense-cored E. chaffeensis, and is found on the surface of the organism and free in the morula space; however, the role of this protein in pathobiology or in eliciting a protective immune response is unknown (Popov et al., 2000). E. chaffeensis p120 has two to five nearly identical serine-rich 80-amino acid TRs, and similarly orthologous E. canis p140 contains 12 or 14 nearly identical serine-rich 36-amino acid TRs (Yabsley et al., 2003; Yu et al., 1997; Yu et al., 2000; Zhang et al., 2008). Specific regions of the p120 and p140 proteins are immunoreactive (McBride et al., 2000; Yu et al., 1996; Yu et al., 2000); however, it is presently unclear as to which sequences within the immunoreactive regions may be recognized by a host immune system.
Current methodologies for diagnosing human monocytotropic ehrlichiosis (HME) present significant clinical limitations. Clinical diagnosis of HME is usually confirmed retrospectively by detection of Ehrlichia-specific antibodies in patient sera using an indirect fluorescent-antibody assay (IFA)(Dumler et al., 2007). The limitations of IFA include lack of standardization between laboratories, false positive interpretations due to autoantibodies or antibodies directed at conserved bacterial proteins, and cross-reactive antibodies produced by related organisms (for example, E. canis, E. ewingii, and Anaplasma phagocytophilum) that can make identification of the specific etiologic agent difficult (Carpenter et al., 1999; Chen et al., 1994; Comer et al., 1999; Paddock and Childs, 2003; Unver et al., 2001). Furthermore, IFA requires expensive microscopy equipment and highly skilled technicians to produce the antigen and interpret results. Molecular diagnostic methods such as PCR are useful for specific and sensitive detection of E. chaffeensis prior to development of reactive antibodies (Childs et al., 1999), but PCR is not useful after antibiotic therapy is initiated, and the clinical sensitivity of PCR in the primary care setting has not been unequivocally determined. Therefore, PCR is currently considered only a valuable adjunct to IFA for diagnosis (Walker et al., 2000). HME diagnosis thus presents significant clinical limitations, and Ehrlichiosis continues to be an emerging infectious disease. Clearly, there is a need for new and improved methods for the detection and diagnosis of Ehrlichiosis.